The recent discovery of RNAi has created the opportunity to develop an entirely new class of drugs against a variety of human diseases that work by silencing disease causing genes. The development of RNAi-based drugs, however, has been hampered by difficulties in delivery and manufacturing, as well as the activation of host interferon-like responses. Of these, delivery has proved to be the major obstacle. RNAi delivery has, thus far, defied conventional approaches that focus mostly on various pharmaceutical technologies, such as siRNA modifications, nanotechnology and liposomes. We hypothesize that non-pathogenic bacteria can be engineered to simultaneously "manufacture" and "deliver" RNAi. In preliminary studies, we show that E. coli or attenuated Salmonella typhimurium expressing shRNA's directed against beta-catenin can induce significant gene silencing in vitro and in vivo. This novel approach offers several advantages. Foremost is clinical safety for bacteria versus viruses since bacteria do not integrate into the human genome. This approach also eliminates the siRNA manufacture issue, and may circumvent or mitigate host interferon-like responses since siRNA is produced intracellularly. The long-term goal of this SBIR proposal is to develop a clinically compatible targeted cancer therapy based on bacterial RNAi. The experiments outlined in this (Phase 1) proposal are necessary for the design and development of bacteria-based RNAi therapy, and are grouped into two specific aims. In Specific Aim 1, we will use various in vitro assays to identify the key mechanisms that regulate bacteria-mediated RNAi, and whether bacterial RNAi is devoid of host interferon-like responses. In Specific Aim 2, we will use two well known models of murine colon cancer to examine whether anaerobic bacteria can be used to selectively deliver shRNA's by taking advantage of the hypoxic milieu inside solid tumors in vivo. Results from these studies are needed to optimize the design and development of our subsequent Phase II studies. Our overall goal in Phase II will be to complete the design and optimization of a clinically compatible RNAi- based targeted therapy for colon cancer using anaerobic bacteria, such as attenuated Salmonella typhimurium. For these studies, attenuated S. typhimurium will be engineered to produce shRNA's directed against the colon cancer oncogene beta-catenin. The ability of the engineered bacteria to mediate therapeutic RNAi in vivo will then be assessed using colon cancer models. The bacteria-based approach, if established, should help to unlock the enormous potential for RNAi-based interventions against a wide spectrum of diseases. PUBLIC HEALTH RELEVANCE: The recent discovery of RNA interference (RNAi), a mechanism that allows specific genes to be }switched off} in cells, holds great promise for the treatment of many diseases but has met with significant problems in terms of delivery. Recently, we have shown that non-pathogenic bacteria can be engineered to activate RNAi in human cells and specifically silence various colon cancer-associated genes. The research outlined in this proposal should enable us to identify the key elements necessary for optimizing bacteria-mediated RNAi, and achieve our overall goal of developing a clinically compatible, bacterial RNAi-based, targeted therapy for colon cancer. [unreadable] [unreadable] [unreadable]